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Immunohistochemistry for
hepatocellular carcinoma
Sanjay Kakar, MD
University of California, San Francisco
2013 Colorado Society of Pathology
Hepatocellular carcinoma
Immunohistochemistry • Commonly used markers:
strengths and limitations
• Different clinical scenarios
Distinction from
• Dysplastic nodule
• Hepatocellular adenoma
HCC immunohistochemistry
Hepatocellular differentiation
• Hep Par 1
• Polyclonal CEA
• Glypican-3
• Arginase-1
• Others: AFP, CD10, villin, TTF-1
Hep Par 1
Strengths Limitations High sensitivity and specificity (>80%)
Negative: 50% of poorly differentiated, scirrhous HCC
Most adenocarcinomas are negative
Focal staining 10-20%
Other polygonal cell tumors often negative
Positive: 20-30% lung, esophageal, gastric adenoCA
Well studied in different tumors
Hep Par 1 in gastric adenocarcinoma
Polyclonal CEA
Strengths Limitations
High sensitivity (>80%) Negative: 50% of poorly differentiated, scirrhous HCC
Canalicular pattern is specific Can be difficult to interpret due to cytoplasmic staining
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HCC Adenocarcinoma
Glypican-3
Strengths Limitations High sensitivity in poorly differentiated, scirrhous HCC (>80%)
Low sensitivity in well (<50%) and moderately differentiated HCC
Negative in adenoma and most high-grade dysplastic nodules
Positive in occasional cirrhotic nodules
Positive in other tumors: yolk sac, melanoma, some adenocarcinomas
Baumhoer, Am J Clin Pathol 2008
Glypican-3 in HCC
GPC-3 Hep Par 1
Shafizadeh/Kakar, Mod Pathol 2009
Glypican-3 in cirrhotic nodule
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Glypican-3 in melanoma Glypican-3 in metastatic breast CA
Arginase-1
Strengths Limitations
High sensitivity (90%), including poorly differentiated, scirrhous HCC
Limited experience
High specificity (>90%): most other tumors are negative
Rare positive staining in other tumors: -Prostatic adenocarcinoma -Cholangiocarcinoma (weak, focal)
Tan, AJSP, 2010
Philips/Kakar, USCAP 2012
Arginase-1: nuclear and/or cytoplasmic
Normal liver HCC
Hepatocellular markers
Well-diff Mod diff Poorly diff
Hep Par 1 100% 83% 46%
Arginase-1 100% 96% 85%
Tan, AJSP, 2010
Sensitivity of commonly used
hepatocellular markers
Well diff Mod diff Poorly diff
Hep Par 1 100% 98% 63%
pCEA 92% 88% 60%
GPC-3 62% 83% 86%
Arginase-1 100% 100% 97%
Philips/Torbenson/Kakar, USCAP 2012
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Arginase
Hep Par 1
Other markers
Marker Limitations
AFP Low sensitivity (30%), background staining
Villin, CD10 Similar to polyclonal CEA
TTF-1 Staining similar to Hep Par 1 Clone-dependent
CD34 Sinusoidal pattern not specific
Albumin in situ hybridization
Not widely available
‘Adenocarcinoma’ markers
Marker
MOC31 (EPCAM)
CK7
CK19
Pan CK (AE1/AE3)
Use
Most adenocarcinomas Neuroendocrine tumors HCC: 5-20%
Positive in most HCCs
Site specific markers TTF-1, napsin A, PSA, ER/PR, GCDFP-15, CDX-2
HCC with MOC31 staining
HCC with CK19 staining HCC: immunohistochemistry
Non-cirrhotic liver Cirrhotic liver
Benign: HCA, FNH High-grade dysplastic nodule
Cholangiocarcinoma Metastatic adenocarcinoma
Cholangiocarcinoma
Polygonal cell tumors: NET, adrenocortical carcinoma, RCC, melanoma, sarcomas
Metastatic tumors: rare
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Needle biopsy for HCC
No stains necessary
• Bile production
• Cirrhotic liver: characteristic features
Trabecular pattern
Fat and/or Mallory hyaline
‘Mesothelioma’ approach
2 hepatocellular markers
2 ‘adenocarcinoma’ markers
Arginase-1 Glypican-3 Hep Par 1
Polyclonal CEA
MOC31 CK19 CK7
Other markers Clinical setting
TTF-1, CDX-2, ER/PR etc If appropriate
2 marker approach Arg-1, MOC31
Limited material
Four groups
Arginase-1 MOC31
Group 1 + -
Group 2 - +
Group 3 + +
Group 4 - -
Arginase+ MOC31 –
• Establishes the diagnosis of HCC in
most situations
• Additional work-up if
-clinical info/morphology not typical
-staining pattern focal
Arginase – MOC31 +
Differential diagnosis
• Adenocarcinoma
• Polygonal cell tumors:
RCC, NE tumor
• HCC (rare)
Arginase+ MOC31+
Differential diagnosis
• MOC31+ HCC
• Adenocarcinoma/NET with
arginase expression (rare)
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Arginase – MOC31 –
Pancytokeratin + Pancytokeratin -
HCC
Adenocarcinoma
NE tumors, RCC
Urothelial CA
Squamous cell CA
Melanoma
Adrenocortical CA
Angiomyolipoma
Sarcomas with
epithelioid pattern
Case 1: 66/M, 6 cm liver mass
no other known tumor
Hep Par 1
IHC summary
• Hep Par 1 +
• pCEA +
• Pan CK +
• CK7 –
• CK20 –
• TTF1 –
Hep Par 1
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Additional stains
Hep Par CK7 Arginase-1 MOC31
+ - - +
• HCC (rare)
• Non-HCC with aberrant Hep Par
Adenocarcinoma
NET
Renal cell carcinoma
Chromogranin
HCC NET Arg-1, GPC-3, Hep Par 1
Positive Hep, GPC-3 rarely positive
MOC31 CK19, CK7
5-20% Usually positive
Chromogranin Synaptophysin CD56
Negative Rare positive Variable
Positive Positive Positive
Immunostaining in HCC and NET
Hep Par 1 GPC-3 Synapto-
physin Chromo-granin A
CD56
HCC (n = 114)
92
67
3 0 7
NET (n = 48)
0 0 47 40 39
Zhou/Frankel, USCAP meeting, 2011
WHO terminology
Term Mitoses/10HPF Ki67 index
Neuroendocrine tumor, grade 1
<2 <2%
Neuroendocrine tumor, grade 2
2-20 3-20%
Neuroendocrine carcinoma, large cell or small cell
>20 >20%
* Mitoses: 13/10 hpf Ki-67 index 50% *
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Diagnosis
Large cell neuroendocrine
carcinoma (grade 3, WHO 2010
grading scheme)
Primary site of NET
Immunohistochemistry Primary site
Comments
TTF-1 Lung Not specific; NETs at other sites can be TTF-1 positive
CDX-2 Intestine Occasional positivity at other sites: pancreatic NET
PAX-8 Pancreas Limited data
Pancreatic NET PAX8 HCC or renal cell carcinoma
Hep Par 1 Hep Par 1
Marker HCC Clear cell RCC Arg-1, GPC-3 Positive Negative
Hep Par 1 Positive Negative
PAX-2 or PAX-8
Negative
Positive Other GU/GYN tumors
RCC marker, EMA, vimentin
Negative Positive
CD10 Canalicular Membranous
Two-stain approach for clear cell tumors:
Arg-1 and PAX-2/PAX-8
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Case 2: 60M, multiple liver and lung masses Initial work-up
Negative
Hep Par 1
pCEA
CK7
TTF-1
PSA
RCC marker
Possible kidney mass
No tissue in the block
PAX-2 nuclear+: metastatic RCC HCC vs. polygonal cell tumors
Polygonal cell tumor Marker
Adrenocortical CA Inhibin
Melan A
Epithelioid
angiomyolipoma
SMA
HMB-45, Melan A
Melanoma SOX10, S-100
HMB-45, Melan A
Arginase, Hep Par 1: negative
GPC-3: melanoma
Case 3: 72/M, tumor nodules in liver,
lung, bones, primary unknown
S-100 SOX-10
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Case 4: 50F, no cirrhosis, 5 cm liver tumor
Negative
Hep Par 1
pCEA
CK7
CK20
Differential diagnosis
• Hep Par 1 negative HCC
• CK7 negative adenocarcinoma
• Polygonal cell tumor:
Neuroendocrine tumor
Renal cell carcinoma
Adrenocortical carcinoma
Melanoma
Angiomyolipoma
Hepatic angiomyolipoma
• Monotypic: lacks ‘angio’ and ‘lipoma’ components
• Myo component is often epithelioid
• Not associated with TS
• IHC: Hep Par 1, MOC31, CK: -ve
Smooth muscle, HMB 45: +ve
*
Round 2
• Arg, GPC-3
• MOC31, CK19
------------------
• CG, Syn
• SOX-10, S-100
• PAX2
• Inhibin
• SMA, HMB-45
• Pan CK
All negative
Arginase – CK19 –
Pan CK + Pan CK -
HCC
Adenocarcinoma
NE tumors, RCC
Urothelial CA
Squamous cell CA
Melanoma
Adrenocortical CA
Angiomyolipoma
Sarcomas with
epithelioid pattern
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Kit: Metastatic GIST Case 5: 55/M with cirrhosis, 6 cm liver mass
Hep Par, pCEA MOC31
Misleading features
• Abundant stroma
• Immunophenotypic features
Negative: Hep Par 1, pCEA
Positive: MOC31
GPC-3 CK19
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Clinicopathologic features
Scirrhous HCC (>50% fibrous stroma)
Conventional HCC
Multinodular 65% 19%
Fibrous capsule 0 71%
Portal tracts 70% 16%
Necrosis 0 70%
Imaging
Peripheral enhancement
62% 3%
Prolonged enhancement
95% 4%
Areas of venous washout
19% 99%
No cirrhosis 15-25% 15-20%
Outcome Better/same/worse
* Immunostain Scirrhous HCC Conventional
HCC
Hep Par 1 17-20% 80-90%
pCEA 33% 60-80%
K7 58-65% 0-20%
K19 50% 0-10%
MOC31 64% 5-11%
Matsuura, Histopath, 2005
Krings/Kakar, Mod Pathol, 2013
Arginase 95% 95%
Glypican-3 95% 70-80%
*
Case 7
• 62 year old woman with a 6 cm
liver mass
• No clinical evidence of chronic
liver disease
*
Hep Par 1 CK19
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Differential diagnosis
• Hepatocellular carcinoma with
pseudoglandular differentiation
• Cholangiocarcinoma with poorly
differentiated component
• Combined hepatocellular-
cholangiocaricnoma
* HCC or CC: clinical impact
HCC CC or Combined HCC-CC
Lymph nodes may not be removed
Lymph node dissection is routine
HCC CC or Combined HCC-CC
Sorafenib, transarterial chemoembolization
Gemcitabine-based or fluoropyramidine-based
HCC CC or Combined HCC-CC
Liver transplant: Milan/UCSF criteria
Likely denial
*
Arginase-1 *
CK19+ HCC stem cell phenotype
• Microvascular invasion
• Fibrous stroma
• Independent predictor of poor
survival
Kim, Hepatology, 2011
*
HCC to CC spectrum: a new classification?
HCC CK19-
HCC CK19+
Scirrhous HCC CK19+
HCC-CC, classical CK19+
CC CK19+
*
Evidence for CC component
Morphology Discrete gland formation Mucin: positive Fibrous stroma
IHC CK19, CK7, MOC31: strong + Arg: negative GPC-3: negative (>90%)
*
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HCC subtypes
HCC subtype Unusual features
Scirrhous HCC Hep, pCEA: often negative Arg-1, GPC: reliable MOC31, CK19: can be +
Fibrolamellar carcinoma
CK7 usually positive Rare: mucin + NE markers + CD68+ may be helpful
Combined HCC-CC CK19+ alone is not enough for diagnosis of CC component Distinct glands, mucin for CC
Summary
Setting Approach
Bile, typical morphology in cirrhosis
No stains
Limited biopsy or Cirrhosis, not typical
2 stain approach: Arginase-1, MOC31
Most situations 4 stain approach: Arginase-1, GPC-3/Hep Par 1 MOC31, CK19
• Avoid large reflex staining panels
• Avoid less useful markers like AFP
• Use site-specific markers judiciously
HCC vs. high grade
dysplastic nodule
Terminology of HCC
Small HCC: <2 cm
High likelihood of cure
• Progressed HCC: Typical features
Nodular HCC
• Early HCC: Resemble HGDN
Vaguely nodular HCC
Stromal invasion
HGDN vs HCC:
clinical implications
High-grade dysplastic nodule
Rebiopsy ?Ablation
Hepatocellular carcinoma
Ablation or resection Priority for transplant
Case 8
• 48 year old male with chronic
hepatitis C and cirrhosis
• 2.5 cm hepatic mass noticed on
screening ultrasound
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Cirrhosis Atypical features Small cell change, pseudoacinar
Reticulin stain High grade DN vs. early HCC
HGDN Early HCC
Small cell change + +
Pseudoglands + +
Trabeculae 1-3 1-3
Portal tracts + +
Unpaired arteries Few Few
Reticulin N or focal N or focal
Stromal invasion - +
Stromal invasion in HCC
• Earliest morphological feature of
HCC
• Invasion of neoplastic cells into
portal tracts, septa, adjacent
parenchyma or blood vessels
Stromal invasion
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Stromal invasion Ductular reaction and
stromal invasion
CK7+ DR at nodular interface
Regenerative Present
HGDN Largely present
HCC Absent or focal
Stromal invasion: no or minimal DR
Park, Cancer, 2007
HCC with stromal invasion CK7+ DR absent in HCC
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HGDN vs early HCC
Stromal invasion
• CK7+ ductular reaction
Immunohistochemistry
• CD34
• Heat shock protein 70 (HSP70)
• Glutamine synthetase (GS)
• Glypican-3
CD34 in HGDN: peripheral
CD 34 in HCC: multifocal/diffuse Immunohistochemistry
• CK7+ ductular reaction
• CD34
• Heat shock protein 70 (HSP70)
• Glutamine synthetase (GS)
• Glypican-3 (GPC-3)
HSP70
Early HCC and non-cancer liver
• 12,600 genes
• HSP70: most abundantly upregulated
in HCC
• Cell cycle progression and apoptosis
Chuma, Hepatology, 2003
HSP70 in HCC: nuclear and cytoplasmic
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HSP70 in adjacent liver: bile duct staining Combined immunostaining
HSP70, GS and GPC-3
Tamasso, Hepatol 07
All negative
Any one + Any two + All positive
HGDN 72% 28% 0 0
HCC 9% 91% 72% 44%
Tamasso, Hepatol 09
All negative
Any one + Any two + All positive
HGDN 78% 22% 0 0
HCC 8% 90% 50% 20%
CD34+ HSP70+ GS+ GPC- Actual practice
Marker Interpretation
HSP70 Often positive in adjacent liver Diagnosis obvious in most cases when positive
GPC-3 Very low sensitivity Rarely helpful
GS Helpful if diffuse staining
Summary
High grade dysplastic
nodule
Early HCC
Stromal invasion Absent Present
CK7+ ductular reaction
Present Absent in area of invasion
CD34 Patchy Multifocal or diffuse
HSP70, GPC, GS <2 >2
Morphology Reticulin
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